This invention relates generally to oligonucleotides and oligonucleotide arrays. More particularly, the invention pertains to arrays of oligonucleotides containing nonhybridizing segments, and to methods for preparing and using such arrays.
Nucleic acid hybridization is a known method for identifying specific sequences of nucleic acids. Hybridization is based upon pairing between complementary nucleic acid strands. Single-stranded oligonucleotides having known sequences can be used as probes to identify target sequences of nucleic acid analytes, by exposing the probes to sample solutions containing nucleic acid analytes of interest. If a nucleic acid analyte hybridizes to a probe, the analyte necessarily contains the target sequence. Various aspects of this method have been studied in detail. In essence, all variations allow complementary base sequences to pair and form double-stranded molecules, and a number of methods are known in the art to determine whether pairing has occurred, such as those described in U.S. Pat. No. 5,622,822 to Ekeze et al. and U.S. Pat. No. 5,256,535 to Ylikoski et al.
High density arrays of oligonucleotides are now known and commercially available for a number of purposes. These so-called xe2x80x9cDNA chipsxe2x80x9d or xe2x80x9cgene chipsxe2x80x9d can be used, for example, in gene expression analysis and mutation detection, polymorphism analysis, mapping, evolutionary studies, and other applications. It has recently become possible to provide single chips that represent the entire human genome. Microfabricated arrays have primarily been manufactured using photolithography, piezoelectric technology technologies, and mechanical spotting. The photolithography approach makes use of semiconductor processing techniques wherein masks are used to selectively irradiate and thus activate modified nucleoside phosphoramidites in the stepwise synthesis of oligonucleotides. See, for example, U.S. Pat. Nos. 5,424,186, 5,445,934, 5,489,678 and 5,744,305 to Fodor et al. and U.S. Pat. No. 5,405,783 to Pirrung et al. With the piezoelectric approach, nucleoside monomers or oligonucleotide segments are delivered to a substrate surface using noncontact inkjet printing technology, i.e., ejected from individual nozzles onto specific sites on a substrate surface; see, for example, U.S. Pat. No. 6,015,880 to Baldeschwieler et al. Microspotting involves direct surface contact wherein microspotting pins, capillaries or tweezers are used to transfer intact biomolecules onto a solid surface.
Each of the aforementioned techniques provides an oligonucleotide array in which the oligonucleotides are intended to serve as hybridization probes, with each oligonucleotide generally intended to bind to at most one complementary oligonucleotide segment. If an array probe were used as a multifunctional probe, i.e., capable of hybridizing to two or more complementary oligonucleotide segments, there would be no means for isolating regions where hybridization is desired from regions where it is not. Thus, known arrays of oligonucleotide probes are limited in this respect.
Accordingly, there is a need in the art for more versatile oligonucleotide arrays wherein the individual oligonucleotides of the array can be used as multifunctional probes, in turn substantially enhancing the utility of a single array.
It is thus a primary object of the invention to address the above-mentioned need in the art by providing partially nonhybridizing oligonucleotides useful as multifunctional probes in an oligonucleotide array.
It is another object of the invention to provide oligonucleotide arrays wherein at least one of the oligonucleotides is a partially nonhybridizing oligonucleotide comprised of two discrete hybridizing segments with a nonhybridizing spacer segment therebetween.
It is another object of the invention to provide oligonucleotide arrays wherein at least one of the oligonucleotides is a partially nonhybridizing oligonucleotide comprised of a plurality of hybridizing oligonucleotide segments, with any two such segments separated by a nonhybridizing spacer segment.
It is still another object of the invention to provide such arrays wherein the nonhybridizing spacer segment is a nucleotidic or nonnucleotidic segment that has little or no likelihood of binding to an oligonucleotide sequence found in nature.
It is yet another object of the invention to provide a method for fabricated oligonucleotides containing one or more partially nonhybridizing oligonucleotides as above, the method comprising attaching pre-synthesized oligonucleotides to designated sites on a substrate surface.
It is a further object of the invention to provide a method for fabricated oligonucleotides containing one or more partially nonhybridizing oligonucleotides as above, the method comprising successively coupling individual nucleotidic monomers and/or oligonucleotide segments to a each of a plurality of designated sites on a substrate surface.
It is an additional object of the invention to provide such a method wherein the pre-synthesized oligonucleotides, nucleotidic monomers and/or oligonucleotide segments are applied to the designated sites on a substrate surface using focused acoustic energy.
It is still an additional object of the invention to provide a method of using an array of oligonucleotides as described herein in a hybridization assay.
Additional objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention.
In one aspect of the invention, then, an oligonucleotide array is provided that is comprised of a plurality of oligonucleotides attached to a substrate surface, wherein at least one of the oligonucleotides is a partially nonhybridizing oligonucleotide comprised of two or more discrete hybridizing segments with a nonhybridizing spacer segment separating any two hybridizing segments. The hybridizing segments are generally oligonucleotide segments approximately 8 to 400 nucleotides in length, preferably in the range of approximately 16 to 80 nucleotides in length. The nonhybridizing spacer segments may be nucleotidic or nonnucleotidic, although nucleotidic spacer segments are preferred. Nucleotidic spacer segments include, by way of example, oligomeric segments comprised of a recurring single nucleotide, e.g., (A)n, (T)n, (G)n or (C)n wherein n is an integer in the range of about 9 to 50, preferably about 9 to 20. Within any given partially nonhybridizing oligonucleotide, the hybridizing segments may be the same or different; in either case, it is possible to use a single partially nonhybridizing oligonucleotide as a multifunctional probe by virtue of containing more than one hybridizing oligonucleotide segment.
In another aspect, a multilayer oligonucleotide array is provided comprised of a plurality of identical partially nonhybridizing oligonucleotides, each of which has the formula
SS-(L)m-X1-Y1-X2-(Y2-X3)n
wherein SS is a substrate surface, L is a linking moiety, X1, X2 and X3 are hybridizing oligonucleotide segments, Y1 and Y2 are nonhybridizing spacer segments, m is zero or 1, and n is an integer in the range of zero to 50, wherein each nonhybridizing spacer segment Yi of each oligonucleotide is covalently linked to the corresponding nonhybridizing spacer segment Yi of at least one adjacent oligonucleotide through a linking moiety containing a cleavable site, and further wherein with any one partially nonhybridizing oligonucleotide, (a) the hybridizing segments may be the same or different, and (b) if more than one nonhybridizing segment is present, the nonhybridizing segments may be the same or different. In this way, a ladder-type structure is provided with two or more layers of hybridizing array segments, whereby the upper array may be used first and the uppermost linking moiety then cleaved to expose the next array, and so forth. Each xe2x80x9carrayxe2x80x9d layer may the same as the others, or each array layer may be different.
Methods for using these oligonucleotide arrays in hybridization assays are also provided. By virtue of the partially nonhybridizing oligonucleotide probes, each of which acts as a multifunctional probe, the oligonucleotide arrays have substantially enhanced utility and versatility in any of a variety of hybridization assay formats.
The invention also provides a method for fabricated oligonucleotide arrays containing at least one partially nonhybridizing oligonucleotide as described above. The method may comprise xe2x80x9cspottingxe2x80x9d intact oligonucleotides onto designated sites of a substrate surface, or it may comprise in situ synthesis of oligonucleotides by individually coupling individual nucleotidic monomers and/or oligonucleotide segments (e.g., the nonhybridizing segments) at designated sites on a substrate surface. A particularly preferred fabrication method involves the use of focused acoustic energy, wherein an acoustic ejector is used to eject single, extremely small droplets of nucleotide-containing or oligonucleotide-containing fluids from fluid reservoirs (e.g., individual wells in a well plate) onto designated sites of a substrate surface.